PROJECT SUMMARY Duplication of the genome is essential to all cells. Yet very few labs have succeeded in reconstituting the enzymology of the eukaryotic replication fork from pure proteins for mechanistic studies. This is in part due to the numerous proteins required to drive replication and the difficulty in obtaining these many factors. Unlike bacteria, eukaryotes use different DNA polymerases to duplicate the leading and lagging strands, the eukaryotic helicase contains 11 distinct proteins, and there exist numerous eukaryotic replication proteins that have no homologue in bacteria. We have made several breakthrough studies using pure proteins to reconstitute the eukaryotic budding yeast (Saccharomyces cerevisiae) replisome (>30 different subunits). We have determined the organization of replisome proteins by EM, the mechanisms by which DNA polymerase (Pol) ? is directed to the leading strand and Pol ? is directed to the lagging strand, along with quality control mechanisms that prevent these Pols from working on the ?wrong? strands. We also solved the orientation of the CMG helicase while translocating on DNA by cryoEM and the consequent profound implications for origin initiation. Questions to be addressed in this proposal include development of the Homo sapiens (H.s.) replisome machinery to address how metazoan replisomes function to perform fork regression, a genome stability process in higher eukaryotes. Thus far we have purified the difficult multisubunit recombinant H.s. factors, including 11-subunit H.s. CMG helicase and have reconstituted the H.s. leading strand replisome. We will characterize how the H.s. replisome functions, compare it to budding yeast and then examine metazoan specific ATPase fork remodelers that reverse stalled forks for genomic integrity. Fork remodelers have not been studied with replisome proteins, and we will address their action in the presence of H.s. replisome proteins and also the fork protection factors, H.s. RAD51 recombinase and the BRCA2 tumor suppressor. We will also examine how reversed forks with bound BRCA2- RAD51, are restored to enable replication restart. We also propose to continue our understanding of replisome structure through cryoEM of various replisome subcomplexes in both the human and budding yeast systems. In overview, the studies proposed here will provide a deep understanding of the workings of the eukaryotic DNA replication machinery, and its intimate involvement in DNA repair, mutagenesis, and human disease. Replication is also crucial for a positive response to many anticancer drugs that are currently in use. Hence detailed knowledge of this central and vital process to cellular life will provide important information useful to prevention and cure of human disease.